Autoantibody enhanced immunoassays and kits

ABSTRACT

The present disclosure provides immunoassays and kits for detection or quantification of an analyte of interest in a test sample that potentially contains endogenously produced autoantibodies reactive with the analyte

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of allowed U.S. patentapplication Ser. No. 13/450,832 filed Apr. 19, 2012 which is adivisional of U.S. Pat. No. 8,183,002 issued on May 22, 2012, both ofwhich are hereby incorporated in their entirety by reference.

TECHNICAL FIELD

The present disclosure relates to immunoassays and kits for detecting ananalyte of interest in a test sample, and in particular to methods andkits for detecting an analyte in a human test sample that may containendogenous anti-analyte antibodies.

BACKGROUND

Immunoassay techniques have been known for the last few decades and arenow commonly used in medicine for a wide variety of diagnostic purposesto detect target analytes in a biological sample. Immunoassays exploitthe highly specific binding of an antibody to its corresponding antigen,wherein the antigen is the target analyte. Typically, quantification ofeither the antibody or antigen is achieved through some form of labelingsuch as radio- or fluorescence-labeling. Sandwich immunoassays involvebinding the target analyte in the sample to the antibody site (which isfrequently bound to a solid support), binding labeled antibody to thecaptured analyte, and then measuring the amount of bound labeledantibody, wherein the label generates a signal proportional to theconcentration of the target analyte inasmuch as labeled antibody doesnot bind unless the analyte is present in the sample.

A problem with this general approach is that many patients havecirculating endogenous antibodies, or “autoantibodies” against ananalyte of clinical interest. For example, autoantibodies have beendescribed for cardiac troponin, myeloperoxidase (MPO), prostate specificantigen (PSA), and thyroid stimulating hormone (TSH), and otherclinically significant analytes. Autoantibodies create interference intypical sandwich immunoassays that are composed of two or moreanalyte-specific antibodies. For example, cardiac troponin-reactiveautoantibodies may interfere with the measurement of cTnI usingconventional midfragment-specific immunoassays. Thus, interference fromautoantibodies can produce erroneous results, particularly near thecut-off values established for clinical diagnoses, and increases therisk of false negative diagnostic results and the risk that individualswill not obtain a timely diagnosis.

One approach to addressing this problem is to choose analyte-specificantibodies that bind to specific epitopes distinct from the analyteepitopes that react with the autoantibodies. Following this generalapproach, efforts have focused on exploring the use of thousands ofdifferent combinations of two, three and even four analyte-specificantibodies to avoid interference from autoantibodies. However, thiseffort has been largely unsuccessful. It is now evident thatautoantibodies against complex protein analytes are likely to bepolyclonal within a particular sample, and may be even more diverseamong samples from different individuals. Interference from diversepolyclonal autoantibodies may explain the observation that as little as25% or even less of an analyte protein sequence binds toanalyte-specific antibodies, which may in turn explain the lack ofsuccess using this approach.

A need exists in the art for new immunoassay methods that compensate forinterference by autoantibodies in a sample, and in particular for suchmethods that do so without involving redesign of the analyte detectionor capture antibodies.

SUMMARY

In one aspect, the present disclosure relates to an immunoassay fordetecting an analyte of interest in a test sample, the immunoassaycomprising the steps of:

(a) contacting a test sample suspected of containing an analyte ofinterest with a first antibody that binds to at least one epitope on theanalyte of interest to form a first antibody-analyte complex, whereinthe first antibody is immobilized on a solid phase, and further whereinat least one autoantibody in the test sample binds to at least oneepitope on the analyte of interest to form an autoantibody-analytecomplex, wherein said autoantibody binds to the solid phase;

(b) contacting said mixture comprising a first antibody-analyte complexand an autoantibody-analyte complex with a second antibody to form ameasurable assembly comprising a first antibody-analyte-second antibodycomplex and an autoantibody-analyte-second antibody complex; wherein thesecond antibody binds to at least one epitope on the analyte ofinterest, and further wherein, an optical, electrical, orchange-of-state signal of the assembly is measured.

In the above immunoassay, the second antibody can be conjugated to adetectable label, wherein the detectable label is an enzyme,oligonucleotide, nanoparticle chemiluminophore, fluorophore,fluorescence quencher, chemiluminescence quencher, or biotin.

In the above immunoassay, an optical signal can be measured as ananalyte concentration-dependent change in chemiluminescence,fluorescence, phosphorescence, electrochemiluminescence, ultravioletabsorption, visible absorption, infrared absorption, refraction, surfaceplasmon resonance.

In the above immunoassay, the electrical signal can be measured as ananalyte concentration dependent change in current, resistance,potential, mass to charge ratio, or ion count.

In the above immunoassay, the change-of-state signal can be measured asan analyte concentration dependent change in size, solubility, mass, orresonance.

In another aspect, the present disclosure relates to an immunoassay fordetecting an analyte of interest in a test sample, the immunoassaycomprising the steps of:

(a) contacting a test sample suspected of containing an analyte ofinterest with a first antibody that binds to at least one epitope on theanalyte of interest to form a first antibody-analyte complex, whereinthe first antibody is immobilized on a solid phase, and further whereinat least one autoantibody in the test sample binds to at least oneepitope on the analyte of interest to form an autoantibody-analytecomplex, wherein said autoantibody binds to the solid phase;

(b) contacting said mixture comprising a first antibody-analyte complexand an autoantibody-analyte complex, with a second antibody that hasbeen conjugated to a detectable label to form a firstantibody-analyte-second antibody complex and anautoantibody-analyte-second antibody complex; wherein the secondantibody binds to at least one epitope on the analyte of interest andfurther wherein, the detectable label is at least one acridiniumcompound;

(c) generating or providing a source of hydrogen peroxide to the mixtureof step (b);

(d) adding a basic solution to the mixture of step (c) to generate alight signal; and

(e) measuring the light signal generated by or emitted in step (d) anddetecting the analyte of interest in the test sample.

In the above immunoassay, the analyte of interest can be a cardiactroponin, thyroid stimulating hormone (TSH), beta human chorionicgonadotropin (beta-HCG); myeloperoxidase (MPO), prostate specificantigen (PSA), human B-type natriuretic peptide (BNP), myosin lightchain 2, myosin-6 or myosin-7.

In the above immunoassay, the test sample can be whole blood, serum, orplasma.

In the above immunoassay, any acridinium compound can be used. Forexample, the acridinium compound can be an acridinium-9-carboxamidehaving a structure according to formula I:

wherein R1 and R2 are each independently selected from the groupconsisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,carboxyalkyl and oxoalkyl, and

wherein R3 through R15 are each independently selected from the groupconsisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and

optionally, if present, X^(Θ) is an anion.

Alternatively, the acridinium compound is an acridinium-9-carboxylatearyl ester having a structure according to formula II:

wherein R1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, carboxyalkyland oxoalkyl; and

wherein R3 through R15 are each independently selected from the groupconsisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and

optionally, if present, X^(Θ) is an anion.

In the above immunoassay, the solid phase can be selected from the groupconsisting of a magnetic particle, bead, test tube, microtiter plate,cuvette, membrane, a scaffolding molecule, quartz crystal, film, filterpaper, disc and chip.

In the above immunoassay, the first antibody can be selected from thegroup consisting of a polyclonal antibody, a monoclonal antibody, achimeric antibody, a human antibody, and an affinity maturated antibody.

In the above immunoassay the second antibody can be selected from thegroup consisting of a polyclonal antibody, a monoclonal antibody, achimeric antibody, a human antibody, and an affinity maturated antibody.

In the above immunoassay the hydrogen peroxide can be provided by addinga buffer or a solution containing hydrogen peroxide.

In the above immunoassay, the hydrogen peroxide can be generated byadding a hydrogen peroxide generating enzyme to the test sample. Thehydrogen peroxide generating enzyme can be selected for example from thegroup consisting of: (R)-6-hydroxynicotine oxidase, (S)-2-hydroxy acidoxidase, (S)-6-hydroxynicotine oxidase, 3-aci-nitropropanoate oxidase,3-hydroxyanthranilate oxidase, 4-hydroxymandelate oxidase,6-hydroxynicotinate dehydrogenase, abscisic-aldehyde oxidase, acyl-CoAoxidase, alcohol oxidase, aldehyde oxidase, amine oxidase, amine oxidase(copper-containing), amine oxidase (flavin-containing), aryl-alcoholoxidase, aryl-aldehyde oxidase, catechol oxidase, cholesterol oxidase,choline oxidase, columbamine oxidase, cyclohexylamine oxidase,cytochrome c oxidase, D-amino-acid oxidase, D-arabinono-1,4-lactoneoxidase, D-arabinono-1,4-lactone oxidase, D-aspartate oxidase,D-glutamate oxidase, D-glutamate (D-aspartate) oxidase,dihydrobenzophenanthridine oxidase, dihydroorotate oxidase,dihydrouracil oxidase, dimethylglycine oxidase, D-mannitol oxidase,ecdysone oxidase, ethanolamine oxidase, galactose oxidase, glucoseoxidase, glutathione oxidase, glycerol-3-phosphate oxidase, glycineoxidase, glyoxylate oxidase, hexose oxidase, hydroxyphytanate oxidase,indole-3-acetaldehyde oxidase, lactic acid oxidase, L-amino-acidoxidase, L-aspartate oxidase, L-galactonolactone oxidase, L-glutamateoxidase, L-gulonolactone oxidase, L-lysine 6-oxidase, L-lysine oxidase,long-chain-alcohol oxidase, L-pipecolate oxidase, L-sorbose oxidase,malate oxidase, methanethiol oxidase, monoamino acid oxidase,N6-methyl-lysine oxidase, N-acylhexosamine oxidase, NAD(P)H oxidase,nitroalkane oxidase, N-methyl-L-amino-acid oxidase, nucleoside oxidase,oxalate oxidase, polyamine oxidase, polyphenol oxidase,polyvinyl-alcohol oxidase, prenylcysteine oxidase, protein-lysine6-oxidase, putrescine oxidase, pyranose oxidase, pyridoxal 5′-phosphatesynthase, pyridoxine 4-oxidase, pyrroloquinoline-quinone synthase,pyruvate oxidase, pyruvate oxidase (CoA-acetylating), reticulineoxidase, retinal oxidase, rifamycin-B oxidase, sarcosine oxidase,secondary-alcohol oxidase, sulfite oxidase, superoxide dismutase,superoxide reductase, tetrahydroberberine oxidase, thiamine oxidase,tryptophan α,β-oxidase, urate oxidase (uricase, uric acid oxidase),vanillyl-alcohol oxidase, xanthine oxidase, oxidase and combinationsthereof.

In the above immunoassay, the basic solution can be a solution having apH of at least about 10.

Optionally, the above immunoassay may further comprise the step ofquantifying the amount of the analyte of interest in the test sample byrelating the amount of light signal in step (e) to the amount of theanalyte of interest in the test sample either by use of a standard curvefor the analyte of interest or by comparison to a reference standard.The immunoassay may be adapted for use in an automated system orsemi-automated system.

In another aspect, the present disclosure relates to a kit for detectingor quantifying an analyte of interest in a test sample, the kitcomprising a solid phase capable of binding autoantibodies present inthe test sample; a first antibody that binds to at least one epitope onthe analyte of interest, the first antibody bound to the solid phase; asecond antibody that binds to at least one epitope on the analyte ofinterest; and instructions for detecting or quantifying the analyte ofinterest.

In the above kit, a detectable label can be conjugated to the secondantibody. The detectable label can be an enzyme, oligonucleotide,nanoparticle chemiluminophore, fluorophore, fluorescence quencher,chemiluminescence quencher, or biotin. In certain embodiments, thedetectable label is an acridinium compound. The acridinium compound canbe an acridinium-9-carboxamide having a structure according to formulaI:

wherein R1 and R2 are each independently selected from the groupconsisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,carboxyalkyl and oxoalkyl, and

wherein R3 through R15 are each independently selected from the groupconsisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, ifpresent, X^(Θ) is an anion. Alternatively, the acridinium compound canbe an acridinium-9-carboxylate aryl ester having a structure accordingto formula

wherein R1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,carboxyalkyl and oxoalkyl; and

wherein R3 through R15 are each independently selected from the groupconsisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and

optionally, if present, X^(Θ) is an anion. The above kit can furtherinclude a basic solution, such as a solution having a pH of at leastabout 10.

The above kit may further include a hydrogen peroxide source, which canbe a buffer, a solution containing hydrogen peroxide, or a hydrogenperoxide generating enzyme. In kits containing a hydrogen peroxidegenerating enzyme, the enzyme can be selected from the group consistingof: (R)-6-hydroxynicotine oxidase, (S)-2-hydroxy acid oxidase,(S)-6-hydroxynicotine oxidase, 3-aci-nitropropanoate oxidase,3-hydroxyanthranilate oxidase, 4-hydroxymandelate oxidase,6-hydroxynicotinate dehydrogenase, abscisic-aldehyde oxidase, acyl-CoAoxidase, alcohol oxidase, aldehyde oxidase, amine oxidase, amine oxidase(copper-containing), amine oxidase (flavin-containing), aryl-alcoholoxidase, aryl-aldehyde oxidase, catechol oxidase, cholesterol oxidase,choline oxidase, columbamine oxidase, cyclohexylamine oxidase,cytochrome c oxidase, D-amino-acid oxidase, D-arabinono-1,4-lactoneoxidase, D-arabinono-1,4-lactone oxidase, D-aspartate oxidase,D-glutamate oxidase, D-glutamate (D-aspartate) oxidase,dihydrobenzaphenanthridine oxidase, dihydroorotate oxidase,dihydrouracil oxidase, dimethylglycine oxidase, D-mannitol oxidase,ecdysone oxidase, ethanolamine oxidase, galactose oxidase, glucoseoxidase, glutathione oxidase, glycerol-3-phosphate oxidase, glycineoxidase, glyoxylate oxidase, hexose oxidase, hydroxyphytanate oxidase,indole-3-acetaldehyde oxidase, lactic acid oxidase, L-amino-acidoxidase, L-aspartate oxidase, L-galactonolactone oxidase, L-glutamateoxidase, L-gulonolactone oxidase, L-lysine 6-oxidase, oxidase,long-chain-alcohol oxidase, L-pipecolate oxidase, L-sorbose oxidase,malate oxidase, methanethiol oxidase, monoamino acid oxidase,N6-methyl-lysine oxidase, N-acylhexosamine oxidase, NAD(P)H oxidase,nitroalkane oxidase, N-methyl-L-amino-acid oxidase, nucleoside oxidase,oxalate oxidase, polyamine oxidase, polyphenol oxidase,polyvinyl-alcohol oxidase, prenylcysteine oxidase, protein-lysine6-oxidase, putrescine oxidase, pyranose oxidase, pyridoxal 5′-phosphatesynthase, pyridoxine 4-oxidase, pyrroloquinoline-quinone synthase,pyruvate oxidase, pyruvate oxidase (CoA-acetylating), reticulineoxidase, retinal oxidase, rifamycin-B oxidase, sarcosine oxidase,secondary-alcohol oxidase, sulfite oxidase, superoxide dismutase,superoxide reductase, tetrahydroberberine oxidase, thiamine oxidase,tryptophan α,β-oxidase, urate oxidase (uricase, uric acid oxidase),vanillyl-alcohol oxidase, xanthine oxidase, xylitol oxidase andcombinations thereof.

In the above it, the solid phase can be selected from the groupconsisting of a magnetic particle, a bead, a test tube, a microtiterplate, a cuvette, a membrane, a scaffolding molecule, a quartz crystal,a film, a filter paper, a disc and a chip.

In the above it, the first antibody and the second antibody can eachbind to an epitope on analyte of interest selected from the groupconsisting of cardiac troponin, thyroid stimulating hormone (TSH), betahuman chorionic gonadotropin (beta-HCG); myeloperoxidase (MPO), prostatespecific antigen (PSA), human B-type natriuretic peptide (BNP), myosintight chain 2, myosin-6 and myosin-7.

In another aspect, the present disclosure relates to an immunodetectioncomposition including a first detection complex comprising a firstantibody reactive with an analyte of interest and bound to a solidphase, the analyte of interest, and a second antibody reactive with theanalyte of interest, wherein the second antibody has a detectable label,and a second detection complex comprising an autoantibody reactive withthe analyte of interest and bound to the solid phase, the analyte ofinterest, and the second antibody, wherein the first and secondcomplexes generate a measurable optical, electrical, or change-of-statesignal from the detectable label. In the above immunodetectioncomposition, the first detection complex can be bound to the seconddetection complex on the solid phase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic diagram of a typical immunoassay reactionsequence;

FIG. 2 shows a schematic diagram of an immunoassay reaction sequence inwhich autoantibodies reactive with a target analyte and autoantibodiesunrelated to the target analyte are bound to a solid phase bearing anexogenous capture antibody;

FIG. 3 shows a graph of dose-response of human IgG captured on magneticmicroparticles and conjugated to an analyte-specific IgG; and

FIG. 4 shows a graph of a serial dilution of IgG from human serumcaptured on magnetic microparticles and conjugated to an analytespecific IgG,

DETAILED DESCRIPTION

The present disclosure relates to immunoassay methods and kits fordetecting an analyte of interest in a test sample, and more particularlyto methods and kits for detecting an analyte in a human test sample thatmay contain endogenous anti-analyte antibodies. Specifically, theinventors have discovered an alternative approach to address the problemof autoantibodies in immunoassay detection of clinically significantanalytes in a sample, in which a solid phase that bears ananalyte-specific antibody is also capable of binding autoantibodiesagainst the analyte that may be present in the sample. This assayapproach compensates for the presence of autoantibodies in the samplewithout redesign of the analyte-specific detection antibodies or thecapture antibodies, does not require use of an extra anti-human IgGdetection conjugate, and avoids the need of a second assay to identifyproblematic samples.

A. Definitions

Section headings as used in this section and the entire disclosureherein are not intended to be limiting.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For therecitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range 6-9, the numbers 7 and 8 are contemplated inaddition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitlycontemplated.

a) Acyl (and other chemical structural group definitions)

As used herein, the term “acyl” refers to a —C(O)R_(a) group where R_(a)is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl.Representative examples of acyl include, but are not limited to, formyl,acetyl, cylcohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl,benzylcarbonyl and the like.

As used herein, the term “alkenyl” means a straight or branched chainhydrocarbon containing from 2 to 10 carbons and containing at least onecarbon-carbon double bond formed by the removal of two hydrogens.Representative examples of alkenyl include, but are not limited to,ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl,5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

As used herein, the term “alkyl” means a straight or branched chainhydrocarbon containing from 1 to 10 carbon atoms. Representativeexamples of alkyl include, but are not limited to, methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl.

As used herein, the term “alkyl radical” means any of a series ofunivalent groups of the general formula C_(n)H_(2n+1) derived fromstraight or branched chain hydrocarbons.

As used herein, the term “alkoxy” means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy.

As used herein, the term “alkynyl” means a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms and containing atleast one carbon-carbon triple bond. Representative examples of alkynylinclude, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl,3-butynyl, 2-pentynyl, and 1-butynyl.

As used herein, the term “amido” refers to an ammo group attached to theparent molecular moiety through a carbonyl group (wherein the term“carbonyl group” refers to a —C(O)— group).

As used herein, the term “amino” means —NR_(b)R_(c), wherein R_(b), andR_(c), are independently selected from the group consisting of hydrogen,alkyl and alkylcarbonyl.

As used herein, the term “aralkyl” means an aryl group appended to theparent molecular moiety through an alkyl group, as defined herein.Representative examples of aryl alkyl include, but are not limited to,benzyl, 2-phenylethyl, 3-phenylpropyl, and 2-naphth-2-ylethyl.

As used herein, the term “aryl” means a phenyl group, or a bicyclic ortricyclic fused ring system wherein one or more of the fused rings is aphenyl group. Bicyclic fused ring systems are exemplified by a phenylgroup fused to a cycloalkenyl group, a cycloalkyl group, or anotherphenyl group. Tricyclic fused ring systems are exemplified by a bicyclicfused ring system fused to a cycloalkenyl group, a cycloalkyl group, asdefined herein or another phenyl group. Representative examples of arylinclude, but are not limited to, anthracenyl, azulenyl, fluorenyl,indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl. The arylgroups of the present disclosure can be optionally substituted withone-, two, three, four, or five substituents independently selected fromthe group consisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.

As used herein, the term “carboxy” or “carboxyl” refers to —CO₂H or—CO₂.

As used herein, the term “carboxyalkyl” refers to a —(CH₂)_(n)CO₂H or—(CH₂)_(n)CO₂— group where n is from 1 to 10.

As used herein, the term “cyano” means a —CN group. As used herein, theterm “cycloalkenyl” refers to a non-aromatic cyclic or bicyclic ringsystem having from three to ten carbon atoms and one to three rings,wherein each five-membered ring has one double bond, each six-memberedring has one or two double bonds, each seven- and eight-membered ringhas one to three double bonds, and each nine- to ten-membered ring hasone to four double bonds. Representative examples of cycloalkenyl groupsinclude cyclohexenyl, octahydronaphthalenyl, norbomylenyl, and the like.The cycloalkenyl groups can be optionally substituted with one, two,three, four, or five substituents independently selected from the groupconsisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.

As used herein, the term “cycloalkyl” refers to a saturated monocyclic,bicyclic, or tricyclic hydrocarbon ring system having three to twelvecarbon atoms. Representative examples of cycloalkyl groups includecyclopropyl, cyclopentyl, bicyclo[3.1.1]heptyl, adamantyl, and the like.The cycloalkyl groups of the present disclosure can be optionallysubstituted with one, two, three, four, or five substituentsindependently selected from the group consisting of alkoxy, alkyl,carboxyl, halo, and hydroxyl.

As used herein, the term “cycloalkylalkyl” means a —R_(d)R_(e) groupwhere R_(d) is an alkylene group and R_(e) is cycloalkyl group. Arepresentative example of a cycloalkylalkyl group is cyclohexylmethyland the like.

As used herein, the term “halogen” means a —Cl, —Br, —I or —F; the term“halide” means a binary compound, of which one part is a halogen atomand the other part is an element or radical that is less electronegativethan the halogen, e.g., an alkyl radical.

As used herein, the term “hydroxyl” means an —OH group.

As used herein, the term “nitro” means a —NO₂ group.

As used herein, the term “oxoalkyl” refers to —(CH₂)_(n)C(O)R_(a), whereR_(a) is hydrogen, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyland where n is from 1 to 10.

As used herein, the term “phenylalkyl” means an alkyl group which issubstituted by a phenyl group.

As used herein, the term “sulfo” means a —SO₃H group.

As used herein, the term “sulfoalkyl” refers to a —(CH₂)_(n)SO₃H, or—(CH₂)_(n)SO₃— group where n is from 1 to 10.

b) Anion

As used herein, the term “anion” refers to an anion of an inorganic ororganic acid, such as, but not limited to, hydrochloric acid,hydrobromic acid, sulfuric acid, methane sulfonic acid, formic acid,acetic acid, oxalic acid, succinic acid, tartaric acid, mandelic acid,fumaric acid, lactic acid, citric acid, glutamic acid, aspartic acid,phosphate, trifluoromethansulfonic acid, trifluoroacetic acid andfluorosulfonic acid and any combinations thereof.

c) Antibody

As used herein, the term “antibody” refers to a protein consisting ofone or more polypeptides substantially encoded by immunoglobulin genesor fragments of immunoglobulin genes, and encompasses polyclonalantibodies, monoclonal antibodies, and fragments thereof, as well asmolecules engineered from immunoglobulin gene sequences. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as myriad immunoglobulinvariable region genes. Light chains are classified as either kappa orlambda. Heavy chains are classified as gamma, mu, alpha, delta, orepsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,IgD and IgE, respectively.

d) Hydrogen Peroxide Generating Enzyme

As used herein, the term “hydrogen peroxide generating enzyme” refers toan enzyme that is capable of producing as a reaction product thechemical compound having the molecular formula H₂O₂ i.e. hydrogenperoxide. Non-limiting examples of hydrogen peroxide generating enzymesare listed below in Table 1.

TABLE 1 IUBMB ENZYME PREFERRED ACCEPTED COMMON NAME NOMENCLATURESUBSTRATE (R)-6-hydroxynicotine oxidase EC 1.5.3.6 (R)-6-hydroxynicotine(S)-2-hydroxy acid oxidase EC 1.1.3.15 S)-2-hydroxyacid(S)-6-hydroxynicotine oxidase EC 1.5.3.5 (S)-6-hydroxynicotine3-aci-nitropropanoate oxidase EC 1.7.3.5 3-aci-nitropropanoate3-hydroxyanthranilate oxidase EC 1.10.3.5 3-hydroxyanthranilate4-hydroxymandelate oxidase EC 1.1.3.19 (S)-2-hydroxy-2-(4-hydroxyphenyl)acetate 6-hydroxynicotinate dehydrogenase EC 1.17.3.36-hydroxynicotinate Abscisic-aldehyde oxidase EC 1.2.3.14 abscisicaldehyde acyl-CoA oxidase EC 1.3.3.6 acyl-CoA Alcohol oxidase EC1.1.3.13 a primary alcohol Aldehyde oxidase EC 1.2.3.1 An aldehyde amineoxidase amine oxidase (copper-containing) EC 1.4.3.6 primary monoammes,diamines and histamine amine oxidase (flavin-containing) EC 1.4.3.4 aprimary amine aryl-alcohol oxidase EC 1.1.3.7 an aromatic primaryalcohol (2-naphthyl)methanol 3- methoxybenzyl alcohol aryl-aldehydeoxidase EC 1.2.3.9 An aromatic aldehyde Catechol oxidase EC 1.1.3.14Catechol cholesterol oxidase EC 1.1.3.6 Cholesterol Choline oxidase EC1.1.3.17 Choline columbamine oxidase EC 1.21.3.2 Columbaminecyclohexylamine oxidase EC 1.4.3.12 Cyclohexylamine cytochrome c oxidaseEC 1.9.3.1 D-amino-acid oxidase EC 1.4.3.3 a D-amino acidD-arabinono-1,4-lactone oxidase EC 1.1.3.37 D-arabinono-1,4-lactoneD-arabinono-1,4-lactone oxidase EC 1.1.3.37 D-arabinono-1,4-lactoneD-aspartate oxidase EC 1.4.3.1 D-aspartate D-glutamate oxidase EC1.4.3.7 D-glutamate D-glutamate(D-aspartate) oxidase EC 1.4.3.15D-glutamate dihydrobenzophenanthridine oxidase EC 1.5.3.12dihydrosanguinarine dihydroorotate oxidase EC 1.3.3.1 (S)-dihydroorotatedihydrouracil oxidase EC 1.3.3.7 5,6-dihydrouracil dimethylglycineoxidase EC 1.5.3.10 N,N-dimethylglycine D-mannitol oxidase EC 1.1.3.40Mannitol Ecdysone oxidase EC 1.1.3.16 Ecdysone ethanolamine oxidase EC1.4.3.8 Ethanolamine Galactose oxidase EC 1.1.3.9 D-galactose Glucoseoxidase EC 1.1.3.4 β-D-glucose glutathione oxidase EC 1.8.3.3Glutathione Glycerol-3-phosphate oxidase EC 1.1.3.21 sn-glycerol3-phosphate Glycine oxidase EC 1.4.3.19 Glycine glyoxylate oxidase EC1.2.3.5 Glyoxylate hexose oxidase EC 1.1.3.5 D-glucose, D-galactoseD-mannose maltose lactose cellobiose hydroxyphytanate oxidase EC1.1.3.27 L-2-hydroxyphytanate indole-3-acetaldehyde oxidase EC 1.2.3.7(indol-3-yl)acetaldehyde lactic acid oxidase Lactic acid L-amino-acidoxidase EC 1.4.3.2 an L-amino acid L-aspartate oxidase EC 1.4.3.16L-aspartate L-galactonolactone oxidase EC 1.3.3.12L-galactono-1,4-lactone L-glutamate oxidase EC 1.4.3.11 L-glutamateL-gulonolactone oxidase EC 1.1.3.8 L-gulono-1,4-lactone L-lysine6-oxidase EC 1.4.3.20 L-lysine L-lysine oxidase EC 1.4.3.14 L-lysinelong-chain-alcohol oxidase EC 1.1.3.20 A long-chain-alcohol L-pipecolateoxidase EC 1.5.3.7 L-pipecolate L-sorbose oxidase EC 1.1.3.11 L-sorbosemalate oxidase EC 1.1.3.3 (S)-malate methanethiol oxidase EC 1.8.3.4Methanethiol monoamino acid oxidase N⁶-methyl-lysine oxidase EC 1.5.3.46-N-methyl-L-lysine N-acylhexosamine oxidase EC 1.1.3.29N-acetyl-D-glucosamine N-glycolylglucosamine N-acetylgalactosamineN-acetylmannosamine. NAD(P)H oxidase EC 1.6.3.1 NAD(P)H nitroalkaneoxidase EC 1.7.3.1 a nitroalkane N-methyl-L-amino-acid oxidase EC1.5.3.2 an N-methyl-L-amino acid nucleoside oxidase EC 1.1.3.39Adenosine Oxalate oxidase EC 1.2.3.4 Oxalate polyamine oxidase EC1.5.3.11 1-N-acetylspermine polyphenol oxidase EC 1.14.18.1Polyvinyl-alcohol oxidase EC 1.1.3.30 polyvinyl alcohol prenylcysteineoxidase EC 1.8.3.5 An S-prenyl-L-cysteine Protein-lysine 6-oxidase EC1.4.3.13 peptidyl-L-lysyl-peptide putrescine oxidase EC 1.4.3.10butane-1,4-diamine Pyranose oxidase EC 1.1.3.10 D-glucose D-xyloseL-sorbose D-glucono-1,5-lactone Pyridoxal 5′-phosphate synthase EC1.4.3.5 pyridoxamine 5′-phosphate pyridoxine 4-oxidase EC 1.1.3.12Pyridoxine pyrroloquinoline-quinone synthase EC 1.3.3.116-(2-amino-2-carboxyethyl)- 7,8-dioxo-1,2,3,4,5,6,7,8-octahydroquinoline-2,4- dicarboxylate Pyruvate oxidase EC 1.2.3.3Pyruvate Pyruvate oxidase (CoA-acetylating) EC 1.2.3.6 PyruvateReticuline oxidase EC 1.21.3.3 Reticuline retinal oxidase EC 1.2.3.11Retinal Rifamycin-B oxidase EC 1.10.3.6 rifamycin-B Sarcosine oxidase EC1.5.3.1 Sarcosine secondary-alcohol oxidase EC 1.1.3.18 a secondaryalcohol sulfite oxidase EC 1.8.3.1 Sulfite superoxide dismutase EC1.15.1.1 Superoxide superoxide reductase EC 1.15.1.2 Superoxidetetrahydroberberine oxidase EC 1.3.3.8 (S)-tetrahydro berberine Thiamineoxidase EC 1.1.3.23 Thiamine tryptophan αβ,-oxidase EC 1.3.3.10L-tryptophan urate oxidase (uricase, uric acid oxidase) EC 1.7.3.3 uricacid Vanillyl-alcohol oxidase EC 1.1.3.38 vanillyl alcohol Xanthineoxidase EC 1.17.3.2 Xanthine xylitol oxidase EC 1.1.3.41 Xylitol

e) Autoantibody

As used herein, the phrase “autoantibody” refers to an antibody thatbinds to an analyte that is endogenously produced in the subject inwhich the antibody is produced.

f) Antibody-Analyte Complex

As used herein, the phrase “antibody-analyte complex” refers to acombination of an antibody and an antigen, in which the antigen is ananalyte of interest, and the antibody and antigen are bound by specific,noncovalent interactions between an antigen-combining site on theantibody and an antigen epitope. The antigen may be a protein or othermolecule. The term “autoantibody-analyte complex” encompasses anantibody-analyte complex in which the antibody is an antibody that bindsto an analyte that is endogenously produced in the subject in which theantibody is produced.

g) Detectable Label

As used herein the term “detectable label” refers to any moiety thatgenerates a measurable signal via optical, electrical, or other physicalindication of a change of state of a molecule or molecules coupled tothe moiety. Such physical indicators encompass spectroscopic,photochemical, biochemical, immunochemical, electromagnetic,radiochemical, and chemical means, such as but not limited tofluorescence, chemifluorescence, chemiluminescence, and the like.Preferred detectable labels include acridinium compounds such as anacridinium-9-carboximide having a structure according to Formula I asset forth in section B herein below, and an acridinium-9-carboxylatearyl ester having a structure according to Formula II as also set forthin section B herein below.

h) Subject

As used herein, the terms “subject” and “patient” are usedinterchangeably irrespective of whether the subject has or is currentlyundergoing any form of treatment. As used herein, the terms “subject”and “subjects” refer to any vertebrate, including, but not limited to, amammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep,hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (forexample, a monkey, such as a cynomolgous monkey, chimpanzee, etc) and ahuman). Preferably, the subject is a human.

i) Test Sample

As used herein, the term “test sample” generally refers to a biologicalmaterial being tested for and/or suspected of containing an analyte ofinterest and which may also include autoantibodies to the analyte ofinterest. The biological material may be derived from any biologicalsource but (preferably is a biological fluid likely to contain theanalyte of interest. Examples of biological materials include, but arenot limited to, stool, whole blood, serum, plasma, red blood cells,platelets, interstitial fluid, saliva, ocular lens fluid, cerebralspinal fluid, sweat, urine, ascites fluid, mucous, nasal fluid, sputum,synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid,semen, soil, etc. The test sample may be used directly as obtained fromthe biological source or following a pretreatment to modify thecharacter of the sample. For example, such pretreatment may includepreparing plasma from blood, diluting viscous fluids and so forth.Methods of pretreatment may also involve filtration, precipitation,dilution, distillation, mixing, concentration, inactivation ofinterfering components, the addition of reagents, lysing, etc. If suchmethods of pretreatment are employed with respect to the test sample,such pretreatment methods are such that the analyte of interest remainsin the test sample at a concentration proportional to that in anuntreated test sample (e.g., namely, a test sample that is not subjectedto any such pretreatment method/sf).

B. Immunoassay for Detecting an Analyte of Interest in a Test Samplethat May Contain Autoantibodies

The present disclosure relates to an immunoassay for detecting ananalyte of interest in a test sample in which autoantibodies against theanalyte of interest mayor may not be present. Examples of analytes ofinterest for which autoantibodies have been described include but arenot limited to cardiac troponin, myeloperoxidase (MPO), prostatespecific antigen (PSA), and thyroid stimulating hormone (ISM). It willbe understood that the immunoassays described herein are also applicableto the detection of any other analyte of interest for whichautoantibodies not yet described create the risk of interference forimmunodetection of the analyte.

The immunoassay of the present disclosure involves obtaining a testsample from a subject and then detecting the presence of an analyte ofinterest using immunodetection while compensating for the presence ofany autoantibodies against the analyte that may be present in thesample. This is achieved in part by providing a solid phase, which canbe a solid support, on which a first, capture antibody is immobilized,and which also during the course of the immunoassay binds anyautoantibody that may be present in the sample.

Immunoassay Methods

The immunoassay methods of the present disclosure can be carried out inany of a wide variety of formats. A general review of immunoassays isavailable in METHODS IN CELL BIOLOGY VOLUME 37: ANTIBODIES IN CELLBIOLOGY, Asai, ed. Academic Press, Inc. New York (1993), and BASIC ANDCLINICAL IMMUNOLOGY 7TH EDITION, Stites & Terr, eds. (1991), which areherein incorporated by reference in its entirety. FIG. 1 is a schematicdiagram of a typical heterogeneous sandwich immunoassay employing asolid phase (as a solid support) to which is bound a first (capture)antibody reactive with at least one epitope on the analyte of interest.A second (detection) antibody is also reactive with at least one epitopeon the analyte of interest. As is shown in FIG. 1, the second antibodymay be conjugated to a delectable label (as indicated by the starbursticon) that provides a signal that is measured after the detectionantibody binds to the captured analyte. When a test sample containingthe analyte of interest contacts the first antibody, the first antibodycaptures the analyte. The analyte is contacted with the second antibodyresulting in the formation of an immunodetection complex consisting ofthe first antibody, analyte and second antibody, and the complex isbound to the solid phase. The signal generated by the second (detection)antibody is proportional to the concentration of the analyte asdetermined by the rate of formation (k₁) of the immunodetection complexversus the rate of dissociation of the immunodetection complex (k₂). Ascan be inferred from FIG. 1, autoantibodies, which if present areunpredictable as to exactly where on an analyte they will bind, cansubstantially interfere with binding of the first and/or secondantibody, and thus with the resulting signal.

In contrast to an Immunoassay format as described and illustrated inFIG. 1, immunoassays according to the present disclosure employ a solidphase that bears a first (capture) antibody as in FIG. 1, but also iscapable of binding any autoantibodies that may be present in the testsample, FIG. 2 is a schematic diagram of an immunoassay format accordingto the present disclosure, in which the test sample contains multipleautoantibodies as shown, each reactive with at least one differentepitope on the analyte of interest. The test sample may also containautoantibodies that are unrelated to the analyte (not shown). As shownin FIG. 2, the solid phase captures the analyte via binding of theanalyte to the first antibody, but also by directly bindingautoantibodies that are reactive with the analyte (as well as anyunrelated autoantibodies that are not reactive with the analyte). Theresult under appropriate conditions is formation of an immunodetectioncomplex that includes a first antibody-analyte-second antibody complex,and an autoantibody-analyte-second antibody complex, which generates astronger signal than that produced by the immunodetection complex shownin FIG. 1. In the immunoassay of the present disclosure and as shown inFIG. 2, the signal generated by the second (detection) antibody remainsproportional to the concentration of the analyte as determined by therate of formation (k₃) of the new immunodetection complex versus therate of dissociation of the new immunodetection complex (k₄). In testsamples containing no solid support, but hey do not bind any of theanalyte of interest and the signal indicative of the analyte isunaffected.

Thus, according to the present disclosure, an immunoassay of the presentdisclosure to detect the presence of an analyte of interest is aheterogeneous assay employing a solid phase which can be a solidsupport. The immunoassay can be performed for example by immobilizing afirst antibody on the solid phase, wherein the first antibody is anexogenous capture antibody, i.e. an exogenous antibody that is reactivewith at least one epitope on the analyte of interest. The solid phase isalso capable of binding any endogenous autoantibodies that may bepresent in the sample. Under conditions sufficient for specific bindingof the first antibody to the analyte interest, the test sample suspectedof containing the analyte of interest, and which mayor may not containautoantibodies, is contacted with the first (capture) antibody, thusforming a first antibody-analyte complex. In the case of a test samplecontaining at least one autoantibody against the analyte, theautoantibody binds to the solid phase and also can bind to at least oneepitope on the analyte to form an autoantibody-analyte complex. Amixture thus formed of the first antibody-analyte complex and theautoantibody-analyte complex is contacted with a second, detectionantibody that binds to at least one epitope on the analyte of interest.This step is carried out under conditions sufficient for specificbinding of the second antibody to any of the analyte of interest that ispresent in the test sample. The second antibody binds to the analyte toform an immunodetection complex which forms a measurable assemblyincluding the first antibody-analyte-second antibody complex and theautoantibody-analyte-second antibody complex. By “measurable assembly”is meant a configuration of molecules that when formed generates asignal susceptible to physical detection and/or quantification. Incertain embodiments for example, the second antibody may be labeled witha detectable label. Depending on the detection approach used, anoptical, electrical, or change-of-state signal of the assembly ismeasured.

Although the immunoassay is described above as including a sequence ofsteps for illustrative purposes, the test sample may be contacted withthe first (capture) antibody and the second (detection) antibodysimultaneously or sequentially, in any order. Regardless of the order ofcontact, if autoantibodies are present in the sample, the autoantibodiesbind directly to the solid phase. Only those autoantibodies that arereactive with the analyte of interest form part of the immunodetectioncomplex that contains the analyte bound to the first, capture antibody,any autoantibody reactive with the analyte, and the second, detectionantibody.

In one format of a sandwich immunoassay according to the presentdisclosure, detecting comprises detecting a signal from the solidphase-affixed immunodetection complex which is a measurable assemblyincluding a first antibody-analyte-second antibody complex and anautoantibody-analyte-second antibody complex. In one embodiment, theimmunodetection complex is separated from the solid phase, typically bywashing, and the signal from the bound label is detected. In anotherformat of a sandwich immunoassay according to the present disclosure,the immunodetection complex remains a solid phase-affixed complex, whichis then detected.

Antibodies

In the immunoassays according to the present disclosure, the firstantibody can be a polyclonal antibody, a monoclonal antibody, a chimericantibody, a human antibody, an affinity maturated antibody or anantibody fragment. Similarly, the second antibody can be a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a human antibody,an affinity maturated antibody or an antibody fragment.

While monoclonal antibodies are highly specific to the analyte/antigen,a polyclonal antibody can preferably be used as the capture (first)antibody to immobilize as much of the analyte/antigen as possible. Amonoclonal antibody with inherently higher binding specificity for theanalyte/antigen may then preferably be used as the detection (second)antibody. In any case, the capture and detection antibodies preferablyrecognize two non-overlapping epitopes on the analyte to avoid blockageof, or interference by the capture antibody with the epitope recognizedby the detection antibody. Preferably the capture and detectionantibodies are capable of binding simultaneously to different epitopeson the analyte, each without interfering with the binding of the other.

Polyclonal antibodies are raised by injecting (e.g., subcutaneous orintramuscular injection an immunogen into a suitable non-human mammal(e.g., a mouse or a rabbit). Generally, the immunogen should induceproduction of high titers of antibody with relatively high affinity forthe target antigen.

If desired, the antigen may be conjugated to a carrier protein byconjugation techniques that are well known in the art. Commonly usedcarriers include keyhole limpet hemocyanin (KLH), thyroglobulin, bovineserum albumin (BSA), and tetanus toxoid. The conjugate is then used toimmunize the animal.

The antibodies are then Obtained from blood samples taken from theanimal. The techniques used to produce polyclonal antibodies areextensively described in the literature (see, e.g., Methods ofEnzymology, “Production of Antisera With Small Doses of Immunogen:Multiple Intradermal Injections,” Langone, et al. eds. (Acad. Press,1981)). Polyclonal antibodies produced by the animals can be furtherpurified, for example, by binding to and elution from a matrix to whichthe target antigen is bound. Those of skill in the art will know ofvarious techniques common in the immunology arts for purification and/orconcentration of polyclonal, as well as monoclonal, antibodies (see,e.g., Coligan, et al. (1990 Unit 9, Current Protocols in Immunology,Wiley Interscience).

For many applications, monoclonal antibodies (mAbs) are preferred. Thegeneral method used for production of hybridomas secreting mAbs is wellknown (Kohler and Milstein (1975) Nature, 256:495). Briefly, asdescribed by Kohler and Milstein, the technique entailed isolatinglymphocytes from regional draining lymph nodes of five separate cancerpatients with either melanoma, teratocarcinoma or cancer of the cervix,glioma or lung, (where samples were obtained from surgical specimens),pooling the cells, and fusing the cells with SHFP-1. Hybridomas werescreened for production of antibody that bound to cancer cell lines.Confirmation of specificity among mAbs can be accomplished using routinescreening techniques (such as the enzyme-linked immunosorbent assay, or“ELISA”) to determine the elementary reaction pattern of the mAb ofinterest.

As used herein, the term “antibody” encompasses antigen-binding antibodyfragments, e.g., single chain antibodies (scFv or others), which can beproduced/selected using phage display technology. The ability to expressantibody fragments on the surface of viruses that infect bacteria(bacteriophage or phage) makes it possible to isolate a single bindingantibody fragment, e.g., from a library of greater than 10¹⁰ nonbindingclones. To express antibody fragments on the surface of phage (phagedisplay), an antibody fragment gene is inserted into the gene encoding aphage surface protein (e.g., pIII) and the antibody fragment-pill fusionprotein is displayed on the phage surface (McCafferty et al, (1990)Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137).

Since the antibody fragments on the surface of the phage are functional,phage-bearing antigen-binding antibody fragments can be separated fromnon-binding phage by antigen affinity chromatography (McCafferty et al.(1990) Nature, 348: 552-554). Depending on the affinity of the antibodyfragment, enrichment factors of 20-fold-1,000,000-fold are obtained fora single round of affinity selection. By infecting bacteria with theeluted phage, however, more phage can be grown and subjected to anotherround of selection. In this way, an enrichment of 1000-fold in one roundcan become 1,000,000-fold in two rounds of selection (McCafferty et al,(1990) Nature; 348: 552-554). Thus, even when enrichments are low (Markset (1991) J. Mol. Biol. 222: 581-597), multiple rounds of affinityselection can lead to the isolation of rare phage. Since selection ofthe phage antibody library on antigen results in enrichment, themajority of clones bind antigen after as few as three to four rounds ofselection. Thus only a relatively small number of clones (severalhundred) need to be analyzed for binding to antigen.

Human antibodies can be produced without prior immunization bydisplaying very large and diverse V-gene repertoires on phage (Marks etal. (1991) J. Mol. Biol. 222: 581-597). In one embodiment, natural VHand VL repertoires present in human peripheral blood lymphocytes areisolated from unimmunized donors by PCR. The V-gene repertoires can bespliced together at random using PCR to create a scFv gene repertoirewhich can be cloned into a phage vector to create a library of 30million phage antibodies (Id.). From a single “naive” phage antibodylibrary, binding antibody fragments have been isolated against more than17 different antigens, including haptens, polysaccharides, and proteins(Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al. (1993).Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12:725-734;Clackson et al. (1991) Nature. 352: 624-628). Antibodies have beenproduced against self proteins, including human thyroglobulin,immunoglobulin, tumor necrosis factor, and CEA (Griffiths et al. (1993)EMBO J. 12: 725-734). The antibody fragments are highly specific for theantigen used for selection and have affinities in the 1 nM to 100 nMrange (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Griffiths et al.(1993). EMBO J. 12:725-734). Larger phage antibody libraries result inthe isolation of more antibodies of higher binding affinity to a greaterproportion of antigens.

As those of skill in the art readily appreciate, antibodies can beprepared by any of a number of commercial services (e.g., BerkeleyAntibody Laboratories, Bethyl Laboratories, Anawa, Eurogenetec, etc.),

Solid Phase

The solid phase can be any suitable material with sufficient surfaceaffinity to bind a capture antibody and autoantibodies present in thetest sample. The solid phase can take any of a number of forms, such asa magnetic particle, bead, test tube, microtiter plate, cuvette,membrane, a scaffolding molecule, quartz crystal, film, filter paper,disc or a chip. Useful solid phase materials include: natural polymericcarbohydrates and their synthetically modified, crosslinked, orsubstituted derivatives, such as agar, agarose, cross-linked alginicacid, substituted and cross-linked guar gums, cellulose esters,especially with nitric acid and carboxylic acids, mixed celluloseesters, and cellulose ethers; natural polymers containing nitrogen, suchas proteins and derivatives, including cross-linked or modifiedgelatins; natural hydrocarbon polymers, such as latex and rubber;synthetic polymers, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; inorganic materials such as sulfates or carbonates ofalkaline earth metals and magnesium, including barium sulfate, calciumsulfate, calcium carbonate, silicates of alkali and alkaline earthmetals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer. Alt of these materials maybe used in suitable shapes, such as films, sheets, tubes, particulates,or plates, or they may be coated onto, bonded, or laminated toappropriate inert carriers, such as paper, glass, plastic films,fabrics, or the like. Nitrocellulose has excellent absorption andadsorption qualities for a wide variety of reagents including monoclonalantibodies. Nylon also possesses similar characteristics and also issuitable.

Alternatively, the solid phase can constitute microparticles.Microparticles useful in the present disclosure can be selected by oneskilled in the art from any suitable type of particulate material andinclude those composed of polystyrene, polymethylacrylate,polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile,polycarbonate, or similar materials. Further, the microparticles can bemagnetic or paramagnetic microparticles, so as to facilitatemanipulation of the microparticle within a magnetic field. In anexemplary embodiment the microparticles are carboxylated magneticmicroparticles.

Microparticles can be suspended in the mixture of soluble reagents andtest sample or can be retained and immobilized by a support material. Inthe latter case, the microparticles on or in the support material arenot capable of substantial movement to positions elsewhere within thesupport material. Alternatively, the microparticles can be separatedfrom suspension in the mixture of soluble reagents and test sample bysedimentation or centrifugation. When the microparticles are magnetic orparamagnetic the microparticles can be separated from suspension in themixture of soluble reagents and test sample by a magnetic field.

The methods of the present disclosure can be adapted for use in systemsthat utilize microparticle technology including automated andsemi-automated systems wherein the solid phase comprises amicroparticle. Such systems include those described in pending U.S. App.No. 425,651 and U.S. Pat. No. 5,089,424, which correspond to publishedEPO App. Nos. EP 0 425 633 and EP 0 424 634, respectively, and U.S. Pat.No. 5,006,309.

In particular embodiments, the solid phase includes one or moreelectrodes. Capture antibodies can be affixed, directly or indirectly,to the electrode(s). In one embodiment, for example, capture antibodiescan be affixed to magnetic or paramagnetic microparticles which are thenpositioned in the vicinity of the electrode surface using a magnet.Systems in which one or more electrodes serve as the solid phase areuseful where detection is based on electrochemical interactions.Exemplary systems of this type are described, for example, in U.S. Pat.No. 6,887,714 (issued May 3, 2005). The basic method is describedfurther below with respect to electrochemical detection.

The capture antibody can be attached to the solid phase by adsorption,where it is retained by hydrophobic forces. Alternatively, the surfaceof the solid phase can be activated by chemical processes that causecovalent linkage of the capture antibody to the support.

To change or enhance the intrinsic charge of the solid phase, a chargedsubstance can be coated directly onto the solid phase. Ion captureprocedures for immobilizing an immobilizable reaction complex with anegatively charged polymer, described in U.S. App. No. 150,278,corresponding to EP Publication No. 0326100, and U.S. App. No. 375,029(EP Publication No, 0406473), can be employed according to the presentdisclosure to affect a fast solution-phase immunochemical reaction. Inthese procedures, an immobilizable immune complex is separated from therest of the reaction mixture by ionic interactions between thenegatively charged polyanion/immune complex and the previously treated,positively charged matrix and detected by using any of a number ofsignal-generating systems, including, e.g., chemiluminescent systems, asdescribed in U.S. App. No. 921,979, corresponding to EPO Publication No,0 273,115.

If the solid phase is silicon or glass, the surface must generally beactivated prior to attaching the capture antibody. Activated silanecompounds such as triethoxy amino propyl slime (available from SigmaChemical Co., St. Louis, Mo.), triethoxy vinyl slime (Aldrich ChemicalCo., Milwaukee, Wis.), and (3-mercapto-propyl)-trimethoxy silane (SigmaChemical Co., St. Louis, Mo.) can be used to introduce reactive groupssuch as amino-, vinyl, and thiol, respectively. Such activated surfacescan be used to link the capture directly (in the cases of amino orthiol), or the activated surface can be further reacted with linkerssuch as glutaraldehyde, his succinimidyl) suberate, SPPD 9 succinimidyl3-[2-pyridyldithio]propionate), SMCC(succinimidyl-4-[Nmaleimidomethyl]cyclohexane-1-carboxylate), SLAB(succinimidyl[4iodoacetyl]aminobenzoate) and SMPB (succinimidyl 4-[1maleimidophenyl]butyrate) to separate the capture antibody from thesurface. Vinyl groups can be oxidized to provide a means for covalentattachment. Vinyl groups can also be used as an anchor for thepolymerization of various polymers such as poly-acrylic acid, which canprovide multiple attachment points for specific capture antibodies.Amino groups can be reacted with oxidized dextrans of various molecularweights to provide hydrophilic linkers of different size and capacity.Examples of oxidizable dextrans include Dextran T-40 (molecular weight40,000 daltons), Dextran T-110 (molecular weight 110,000 daltons),Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M(molecular weight 2,000,000 daltons) (all of which are available fromPharmacia, Piscataway, N.J.), or Ficoll (molecular weight 70,000daltons; available from Sigma Chemical Co., St. Louis, Mo.).Additionally, polyelectrolyte interactions can be used to immobilize aspecific capture antibody on a solid phase using techniques andchemistries described U.S. App. No. 150,278, filed Jan. 29, 1988, andU.S. App. No. 375,029, filed Jul. 7, 1989, each of which is incorporatedherein by reference.

Other considerations affecting the choice of solid phase include theability to minimize non-specific binding of labeled entities andcompatibility with the labeling system employed, For, example, solidphases used with fluorescent labels should have sufficiently lowbackground fluorescence to allow signal detection.

Following attachment of a specific capture antibody, the surface of thesolid support may be further treated with materials such as serum,proteins, or other blocking agents to minimize non-specific bindingand/or to promote binding of autoantibodies.

Detection Systems In General

As discussed above, immunoassays according to the present disclosureemploy a second, detection antibody that is analyte-specific. In certainembodiments, the second antibody has a detectable label.

Detectable labels suitable for use in the detection antibodies of thepresent disclosure include any compound or composition having a moietythat is detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, or chemical means. Such labelsinclude, for example, an enzyme, oligonucleotide, nanoparticlechemiluminophore, fluorophore, fluorescence quencher, chemiluminescencequencher, or biotin. Thus for example, in an immunoassay employing anoptical signal, the optical signal is measured as an analyteconcentration dependent change in chemiluminescence, fluorescence,phosphorescence, electrochemiluminescence, ultraviolet absorption,visible absorption, infrared absorption, refraction, surface plasmonresonance. In an immunoassay employing an electrical signal, theelectrical signal is measured as an analyte concentration dependentchange in current, resistance, potential, mass to charge ratio, or ioncount. In an immunoassay employing a change-of-state signal, the changeof state signal is measured as an analyte concentration dependent changein size, solubility, mass, or resonance.

Useful labels according to the present disclosure include magnetic beads(e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas Red,rhodamine, green fluorescent protein) and the like (see, e.g., MolecularProbes, Eugene, Oreg., USA), chemiluminescent compounds such asacridinium (e.g., acridinium-9-carboxamide), phenanthridinium,dioxetanes, luminol and the like, radiolabels (e.g., ³R, ¹²⁵I, ³⁵S, ¹⁴C,or ³²P) catalysts such as enzymes (e.g., horse radish peroxidase,alkaline phosphatase, beta-galactosidase and others commonly used in anELISA), and colorimetric labels such as colloidal gold (e.g., goldparticles in the 0.40-80 nm diameter size range scatter green light withhigh efficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

The label can be attached to the detection antibody prior to, or during,or after contact with the biological sample. So-called “direct labels”are detectable labels that are directly attached to or incorporated intothe detection antibody prior to use in the assay. Direct labels can beattached to or incorporated into the detection antibody by any of anumber of means well known to those of skill in the art.

In contrast, so-called “indirect labels” typically bind to the detectionantibody at some point during the assay. Often, the indirect label bindsto a moiety that is attached to or incorporated into the detection agentprior to use. Thus, for example, a detection antibody can bebiotinylated before use in an assay. During the assay, anavidin-conjugated fluorophore can bind the biotin-bearing detectionagent, to provide a label that is easily detected.

In another example of indirect labeling, polypeptides capable ofspecifically binding immunoglobulin constant regions, such aspolypeptide A or polypeptide G, can also be used as labels for detectionantibodies. These polypeptides are normal constituents of the cell wallsof streptococcal bacteria. They exhibit a strong non-immunogenicreactivity with immunoglobulin constant regions from a variety ofspecies (see, generally Kronval, et al. (1973) J. Immunol., 111:1401-1406, and Akerstrom (1985) J. Immunol., 135: 2589-2542). Suchpolypeptides can thus be labeled and added to the assay mixture, wherethey will bind to the capture and detection antibodies, as well as tothe autoantibodies, labeling all and providing a composite signalattributable to analyte and autoantibody present in the sample.

Some labels useful in the present disclosure may require the use of anadditional reagent(s) to produce a detectable signal. In an ELISA, forexample, an enzyme label (e.g., beta-galactosidase) will require theaddition of a substrate (e.g., X-gal) to produce a detectable signal. Inimmunoassays using an acridinium compound as the direct label, a basicsolution and a source of hydrogen peroxide are added.

Detection Systems—Exemplary Formats

Chemiluminescence Immunoassay: In an exemplary embodiment, achemiluminescent compound is used in the above-described methods as adirect label conjugated to the second, detection antibody. Thechemiluminescent compound can be an acridinium compound. When anacridinium compound is used as the detectable label, then theabove-described method may further include generating or providing asource of hydrogen peroxide to the mixture resulting from contacting thetest sample with the first antibody and the second antibody, and addingat least one basic solution to the mixture to generate a light signal.The light signal generated or emitted by the mixture is then measured todetect the analyte of interest in the test sample.

The source of hydrogen peroxide may be a buffer solution or a solutioncontaining hydrogen peroxide or an enzyme that generates hydrogenperoxide when added to the test sample. The basic solution serves as atrigger solution, and the order in which the at least one basic solutionand detectable label are added is not critical. The basic solution usedin the method is a solution that contains at least one base and that hasa pH greater than or equal to 10, preferably, greater than or equal to12. Examples of basic solutions include, but are not limited to, sodiumhydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide,magnesium hydroxide, sodium carbonate, sodium bicarbonate, calciumhydroxide, calcium carbonate and calcium bicarbonate. The amount ofbasic solution added to the test sample depends on the concentration ofthe basic solution used in the assay. Based on the concentration of thebasic solution used, one skilled in the art could easily determine theamount of basic solution to be used in the method described herein.

In a chemiluminescence immunoassay according to the present disclosureand using an acridinium compound as the detectable label, preferably theacridinium compound is an acridinium-9-carboxamide. Specifically, theacridinium-9-carboxamide has a structure according to formula I:

wherein R¹ and R² are each independently selected from the groupconsisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,carboxyalkyl and oxoalkyl, and

wherein R³ through R¹⁵ are each independently selected from the groupconsisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and further whereinany of the alkyl, alkenyl, alkynyl, aryl or aralkyl may contain one ormore heteroatoms; and

optionally, if present, X^(Θ) is an anion.

Methods for preparing acridinium 9-carboxamides are described inMattingly, P. G. J. Biolumin. Chemilumin., 6, 107-14; (1991); Adamczyk,M.; Chen, Y.-Y., Mattingly, P. G.; Pan, Y. Org. Chem., 63, 5636-5639(1998); Adamczyk, M.; Chen, Y.-Y.; Mattingly, P. G.; Moore, 1. A.;Shreder, K. Tetrahedron, 55, 10899-10914 (1999); Adamczyk, M.;Mattingly, P. G.; Moore, J. A.; Pan, Y. Org. Lett., 1, 779-781 (1999);Adamczyk, M.; Chen, Y.-Y.; Fishpaugh, J. R.; Mattingly, P. G.; Pan, Y.;Shreder, K.; Yu, Z. Bioconjugate Chem., 11, 714-724 (2000); Mattingly,P. G.; Adamczyk, M. In Luminescence Biotechnology: Instruments andApplications; Dyke, K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002);Adamczyk, M.; Mattingly, P. G.; Moore, J. A.; Pan, Y. Org. Lett., 5,3779-378 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524 and 5,783,699(each incorporated herein by reference in their entireties for theirteachings regarding same).

Alternatively, the acridinium compound can be anacridinium-9-carboxylate aryl ester; the acridinium-9-carboxylate arylester can have a structure according to formula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl sulfoalkylcarboxyalkyl and oxoalkyl; and

wherein R³ through R¹⁵ are each independently selected from the groupconsisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and

optionally, if present, X^(Θ) is an anion.

Examples of acridinium-9-carboxylate aryl esters having the aboveformula II that can be used in the present disclosure include, but arenot limited to, 10-methyl-q′ (phenoxycarbonyl)acridinium fluorosulfonate(available from Cayman Chemical, Ann Arbor, Mich.). Methods forpreparing acridinium 9-carboxylate aryl esters are described in McCapra,F., et al., Photochem. Photobiol., 4, 1111-21 (1965); Razavi, Z et al.,Luminescence, 15:245-249 (2000); Razavi, Z et al., Luminescence,15:239-244 (2000); and U.S. Pat. No. 5,241,070 (each incorporated hereinby reference in their entireties for their teachings regarding same).

In addition to the at least one acridinium compound, the indicatorsolution can also contain at least one surfactant. Any surfactant thatwhen dissolved in water, lowers the surface tension of the water andincreases the solubility of organic compounds, can be used in thepresent invention. Examples of surfactants that can be used is one ormore non-ionic or ionic surfactants (e.g., anionic, cationic orzwitterionic surfactants). Examples of non-ionic surfactants that can beused include, but are not limited to, t-octylpheoxypolyethoxyethanol(TRITON X-100, Sigma Aldrich, St. Louis, Mo.), polyoxyethylenesorbitanmonolaurate (Tween 20), nonylphenol polyoxyethylene ether (Nonidet P10),decyldimethylphosphine oxide (APO-10), Cyclohexyl-n-ethyl-β-D-Maltoside,Cyclohexyl-n-hexyl-β-D-Maltoside, Cyclohexyl-n-methyl-β-D-Maltoside,n-Decanoylsucrose, n-Decyl-β-D-glucopyranoside,n-Decyl-β-D-maltopyranoside, n-Decyl-β-D-maltoside, Digitonin,n-Dodecanoyl sucrose, n-Dodecyl-β-D-glucopyranoside,n-Dodecyl-β-D-maltoside, polyoxyethylene (10) dodecyl ether (GenapolC-100), isotridecanol polyglycol ether (Genapol X-80), isotridecanolpolyglycol ether (Genapol X-100), Heptane-1,2,3-triol,n-Heptyl-β-D-glucopyranoside, n-Heptyl-β-D-thioglucopyranoside andcombinations thereof. An example of a ionic surfactant that can be usedinclude, sodium cholate, chenodeoxycholic acid, cholic acid,dehydrocholic acid, docusate sodium, docusate sodium salt, glycocholicacid hydrate, glycodeoxycholic acid monohydrate, glycolithocholic acidethyl ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, dodecylsulfate, calcium propionate, 1-octanesulfonic acid sodium salt, sodium1-butanesulfonate, sodium chenodeoxycholate, sodium cholate hydrate,sodium 1-decanesulfonate, sodium 1-decanesulfonate, sodium deoxycholate,sodium deoxycholate monohydrate, sodium dodecylbenzenesulfonate, sodiumdodecyl sulfite, sodium glycochenodeoxycholate, sodium glycocholatehydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium1-nonanesulfonate, sodium octyle sulfate, sodium pentanesulfonate,sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate hydrate,sodium taurohyodeoxycholate hydrate, sodium tauroursodeoxycholate,taurocholic acid sodium salt hydrate, taurolithocholic acid 3-sulfatedisodium salt, Triton® X-200, Triton® QS-15, Triton® QS-44, Triton®XQS-20, Trizma® dodecyl sulfate, ursodeoxycholic acid,alkyltrimethylammonium bromide, amprolium hydrocholoride, benzalkoniumchloride, benzethonium hydroxide, benzyldimethylhexadecylammoniumchloride, benzyldodecyldimethylammonium bromide, cholinep-toluenesulfonate salt, dimethyldioctadecylammonium bromide,dodecylethyldimethylammonium bromide, dodecyltrimethylammonium bromide,ethylhexadecyldimethylammonium bromide, Ggirard's reagent,hexadecylpyridinium bromide, hexadecylpyridinium chloride monohydrate,hexadecylpyridinium chloride monohydrate, hexadecyltrimethylammoniumbromide, hexadecyltrimethylammonium p-toluenesulfonate,hexadecyltrimethylammonium bromide, hexadecyltrimethylammoniump-toluenesulfonate, Hyamine® 1622, methylbenzethonium chloride,myristyltrimethylammonium bromide, oxyphenonium bromide,N,N′,N′-polyoxyethylene (10)-N-tallow-1,3-diaminopropane,tetraheptylammonium bromide, tetrakis(decyl)ammonium bromide, thonzoniumbromide and Luviquat™ FC370, Luviquat™ HM 552, Luviquat™ HOLD, Luviquat™MS 370, Luviquat™ PQ 11PN and combinations thereof (available from SigmaAldrich, St. Louis, Mo.).

Optionally, the test sample may be treated prior to the addition of anyone or more of the at least one basic solution, hydrogen peroxide sourceand detectable label. Such treatment may include dilution,ultrafiltration, extraction, precipitation, dialysis, chromatography anddigestion. Such treatment may be in addition to and separate from anypretreatment that the test sample may receive or be subjected to asdiscussed previously herein. Moreover, if such treatment methods areemployed with respect to the test sample, such treatment methods aresuch that the analyte of interest remains in the test sample at aconcentration proportional to that in an untreated test sample (e.g.,namely, a test sample that is not subjected to any such treatmentmethod(s)).

As mentioned briefly previously herein, the time and order in which thetest sample, the at least one basic solution, source of hydrogenperoxide and the detectable label are added to form a mixture is notcritical. Additionally, the mixture formed by the at least one basicsolution, hydrogen peroxide source and the detectable label, canoptionally be allowed to incubate for a period of time. For example, themixture can be allowed to incubate for a period of time of from about 1second to about 60 minutes. Specifically, the mixture can be allowed toincubate for a period of from about 1 second to about 18 minutes.

When a chemiluminescent detectable label is used, after the addition ofthe at least one basic solution, hydrogen peroxide source, and thedetectable label to the test sample, a detectable signal, namely, achemiluminescent signal, is generated. The signal generated by themixture is detected for a fixed duration of time. Preferably, themixture is formed and the signal is detected concurrently. The durationof the detection may range from about 0.01 to about 360 seconds, morepreferably from about 0.1 to about 30 seconds, and most preferably fromabout 0.5 to about 5 seconds. Chemiluminescent signals generated can bedetected using routine techniques known to those skilled in the art.

Thus, in a chemiluminescent immunoassay according to the presentdisclosure, a chemiluminescent detectable label is used and added to thetest sample, the chemiluminescent signal generated after the addition ofthe basic solution and the detectable label indicates the presence ofthe analyte of interest in the test sample, which signal can bedetected. The amount or concentration of the analyte of interest in thetest sample can be quantified based on the intensity of the signalgenerated. Specifically, the amount of the analyte of interest containedin a test sample is proportional to the intensity of the signalgenerated. Specifically, the amount of the analyte of interest presentcan be quantified based on comparing the amount of light generated to astandard curve for the analyte of interest or by comparison to areference standard. The standard curve can be generated using serialdilutions or solutions to the analyte of interest of knownconcentration, by mass spectroscopy, gravimetrically and by othertechniques known in the art.

Fluorescence Polarization Immunoassay (FPIA): In an exemplaryembodiment, a fluorescent label is employed in a fluorescencepolarization immunoassay (FPIA) according to the invention. Generally,fluorescent polarization techniques are based on the principle that afluorescent label, when excited by plane-polarized light of acharacteristic wavelength, will emit light at another characteristicwavelength (i.e., fluorescence) that retains a degree of thepolarization relative to the incident light that is inversely related tothe rate of rotation of the label in a given medium. As a consequence ofthis property, a label with constrained rotation, such as one bound toanother solution component with a relatively lower rate of rotation,will retain a relatively greater degree of polarization of emitted lightthan when free in solution.

This technique can be employed in immunoassays according to theinvention, for example, by selecting reagents such that binding of thefluorescently labeled entities forms a complex sufficiently different insize such that a change in the intensity light emitted in a given planecan be detected. For example, when a labeled cardiac troponin antibodyis bound by one or more cardiac troponin antigens captured by thecapture antibody and/or autoantibodies reactive with the cardiactroponin, the resulting complex is sufficiently larger, and its rotationis sufficiently constrained, relative to the free labeled cardiactroponin antibody that binding is easily detected.

Fluorophores useful in FPIA include fluorescein, amino fluorescein,carboxyfluorescein, and the like, preferably 5 and6-aminomethylfluorescein, 5 and 6-aminofluorescein,6-carboxyfluorescein, 5-carboxyfluorescein, thioureafluorescein, andmethoxytriazinolyl-amino fluorescein, and similar fluorescentderivatives. Examples of commercially available automated instrumentswith which fluorescence polarization assays can be conducted include:the IMx system, the TDx system, and TDxFLx system (all available fromAbbott Laboratories, Abbott Park, Ill.).

Scanning Probe Microscopy (SPM): The use of scanning probe microscopy(SPM) for immunoassays also is a technology to which the immunoassaymethods of the present disclosure are easily adaptable. In SPM, inparticular in atomic force microscopy, the capture antibody is affixedto the solid phase that in addition to being capable of bindingautoantibodies, has a surface suitable for scanning. The captureantibody can, for example, be adsorbed to aplastic or metal surface.Alternatively, the capture antibody can be covalently attached to, e.g.,derivatized plastic, metal, silicon, or glass according to methods knownto those of ordinary skill in the art. Following attachment of thecapture antibody, the test sample is contacted with the solid phase, anda scanning probe microscope is used to detect and quantify solidphase-affixed complexes. The use of SPM eliminates the need for labelsthat are typically employed in immunoassay systems. Such a system isdescribed in U.S. App. No. 662,147, which is incorporated herein byreference.

MicroElectroMechanical Systems (MEMS): Immunoassays according to thepresent disclosure can also be carried out using aMicroElectroMechanical System (MEMS). MEMS are microscopic structuresintegrated onto silicon that combine mechanical, optical, and fluidicelements with electronics, allowing convenient detection of an analyteof interest. An exemplary MEMS device suitable for use in the presentdisclosure is the Protiveris' multicantitever array. This array is basedon chemo-mechanical actuation of specially designed siliconmicrocantilevers and subsequent optical detection of the microcantileverdeflections. When coated on one side with a binding partner, amicrocantilever will bend when it is exposed to a solution containingthe complementary molecule. This bending is caused by the change in thesurface energy due to the binding event. Optical detection of the degreeof bending (deflection) allows measurement of the amount ofcomplementary molecule bound to the microcantilever.

Electrochemical Detection Systems: In other embodiments, immunoassaysaccording to the present disclosure are carried out usingelectrochemical detection, the techniques for which are well known tothose skilled in the art. Such electrochemical detection often employsone or more electrodes connected to a device that measures and recordsan electrical current. Such techniques can be realized in a number ofcommercially available devices, such as the I-STAT® (AbbottLaboratories, Abbott Park, Ill.) system, which comprises a hand-heldelectrochemical detection instrument and self-contained assay-specificreagent cartridges. For example, in the present invention, the basictrigger solution could be contained in the self-contained hemoglobinreagent cartridge and upon addition of the test sample, a current wouldbe generated at at least one electrode that is proportional to theamount of hemoglobin in the test sample. A basic procedure forelectrochemical detection has been described for example by Heineman andcoworkers. This entailed immobilization of a primary antibody (Ab,rat-anti mouse IgG), followed by exposure to a sequence of solutionscontaining the antigen (Ag, mouse IgG), the secondary antibodyconjugated to an enzyme label (AP-Ab, rat anti mouse IgG and alkalinephosphatase), and p-aminophenyl phosphate (PAPP). The AP converts PAPPto p-aminophenol (PAP_(R), the “R” is intended to distinguish thereduced form from the oxidized form, PAP_(O), the quinoneimine), whichis electrochemically reversible at potentials that do not interfere withreduction of oxygen and water at 9.0, where AP exhibits optimumactivity. PAP does not cause electrode fouling, unlike phenol whoseprecursor, phenylphosphate, is often used as the enzyme substrate.Although PAP_(R) undergoes air and tight oxidation, these are easilyprevented on small scales and short time frames. Picomole detectionlimits for PAP_(R) and femtogram detection limits for IgG achieved inmicroelectrochemical immunoassays using PAPP volumes ranging from 20 μlto 360 μL have been reported previously. In capillary immunoassays withelectrochemical detection, the lowest detection limit reported thus faris 3000 molecules of mouse IgG using a volume of 70 μL and a 30 min or25 min assay time.

In an exemplary embodiment employing electrochemical detection accordingto the present disclosure, a capture antibody reactive with the analyteof interest can be immobilized on the surface of an electrode which isthe solid phase. The electrode is then contacted with a test samplefrom, e.g., a human. Any analyte in the sample binds to the captureantibody to form a first solid phase-affixed complex. Autoantibodiesalso bind to the surface of the electrode thereby becoming immobilizedon the surface of the electrode. Analyte in the test sample that isunbound by the capture antibody binds to immobilized autoantibodies thatare reactive with the analyte to form a second solid phase-affixedcomplex. These solid phase-affixed complexes are contacted with adetection antibody that is analyte-specific and has a detectable label.Formation of an immunodetection complex including the firstantibody-analyte-second antibody complex plus theautoantibody-analyte-second antibody complex results in generation of asignal by the detectable label, which is then detected.

Various electrochemical detection systems are described in U.S. Pat. No.7,045,364 (issued May 16, 2006; incorporated herein by reference), U.S.Pat. No. 7,045,310 (issued May 16, 2006; incorporated herein byreference), U.S. Pat. No. 6,887,714 (issued May 3, 2005; incorporatedherein by reference), U.S. Pat. No. 6,682,648 (issued Jan. 27, 2004;incorporated herein by reference); U.S. Pat. No. 6,670,115 (issued Dec.30, 2003; incorporated herein by reference).

In the above immunoassay, any acridinium compound can be used. Forexample, the acridinium compound can be an acridinium-9-carboxamidehaving a structure according to formula I:

wherein R1 and R2 are each independently selected from the groupconsisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,carboxyalkyl and oxoalkyl, and

wherein R3 through R15 are each independently selected from the groupconsisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,cyano, sulfo, carboxyalkyl and oxoalkyl; and

optionally, if present, X^(θ) is an anion.

Alternatively, the acridinium compound is an acridinium-9-carboxylatearyl ester having a structure according to formula II:

wherein R1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,carboxyalkyl and oxoalkyl; and

wherein R3 through R15 are each independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, aryl or aralkyl, amino, amido,acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo,sulfoalkyl, carboxyalkyl and oxoalkyl; and

optionally if present, X^(Θ) is an anion.

In the above immunoassay the hydrogen peroxide can be provided by addinga buffer or a solution containing hydrogen peroxide.

In the above immunoassay, the hydrogen peroxide can be generated byadding a hydrogen peroxide generating enzyme to the test sample. Thehydrogen peroxide generating enzyme can be selected for example from thegroup consisting of: (R)-6-hydroxynicotine oxidase, (S)-2-hydroxy acidoxidase, (S)-6-hydroxynicotine oxidase, 3-aci-nitropropanoate oxidase,3-hydroxyanthranilate oxidase, 4-hydroxymandelate oxidase,6-hydroxynicotinate dehydrogenase, abscisic-aldehyde oxidase, acyl-CoAoxidase, alcohol oxidase, aldehyde oxidase, amine oxidase, amine oxidase(copper-containing), amine oxidase (flavin-containing), aryl-alcoholoxidase, aryl-aldehyde oxidase, catechol oxidase, cholesterol oxidase,choline oxidase, columbamine oxidase, cyclohexylamine oxidase,cytochrome c oxidase, D-amino-acid oxidase, D-arabinono-1,4-lactoneoxidase, D-arabinono-1,4-lactone oxidase, D-aspartate oxidase,D-glutamate oxidase, D-glutamate (D-aspartate) oxidase,dihydrobenzophenanthridine oxidase, dihydroorotate oxidase,dihydrouracil oxidase, dimethylglycine oxidase, D-mannitol oxidase,ecdysone oxidase, ethanolamine oxidase, galactose oxidase, glucoseoxidase, glutathione oxidase, glycerol-3-phosphate oxidase, glycineoxidase, glyoxylate oxidase, hexose oxidase, hydroxyphytanate oxidase,indole-3-acetaldehyde oxidase, lactic acid oxidase, L-amino-acidoxidase, L-aspartate oxidase, L-galactonolactone oxidase, L-glutamateoxidase, L-gulonolactone oxidase, L-lysine 6-oxidase, oxidase,long-chain-alcohol oxidase, L-pipecolate oxidase, L-sorbose oxidase,malate oxidase, methanethiol oxidase, monoamino acid oxidase,N6-methyl-lysine oxidase, N-acylhexosamine oxidase, NAD(P)H oxidase,nitroalkane oxidase, amino-acid oxidase, nucleoside oxidase, oxalateoxidase, polyamine oxidase, polyphenol oxidase, polyvinyl-alcoholoxidase, prenylcysteine oxidase, protein-lysine 6-oxidase, putrescineoxidase, pyranose oxidase, pyridoxal 5′-phosphate synthase, pyridoxine4-oxidase, pyrroloquinoline-quinone synthase, pyruvate oxidase, pyruvateoxidase (CoA-acetylating), reticuline oxidase, retinal oxidase,rifamycin-B oxidase, sarcosine oxidase, secondary-alcohol oxidase,sulfite oxidase, superoxide dismutase, superoxide reductase,tetrahydroberberine oxidase, thiamine oxidase, tryptophan α,β-oxidase,urate oxidase (uricase, uric acid oxidase), vanillyl-alcohol oxidase,xanthine oxidase, oxidase and combinations thereof.

C. Kits

The present disclosure also provides kits for assaying test samples forpresence of an analyte of interest wherein the test sample may containautoantibodies. Kits according to the present disclosure include one ormore reagents useful for practicing one or more immunoassays accordingto the present disclosure. A kit generally includes a package with oneor more containers holding the reagents, as one or more separatecompositions or, optionally, as admixture where the compatibility of thereagents will allow. The test kit can also include other material(s),which may be desirable from a user standpoint, such as a buffer(s),diluent(s), a standard(s), and/or any other material useful in sampleprocessing, washing, or conducting any other step of the assay.

In certain embodiments, a test kit includes a humanized monoclonalantibody, wherein the humanized monoclonal antibody is specific for theanalyte of interest. This component can be used as a positive control inimmunoassays according to the invention. If desired, this component canbe included in the test kit in multiple concentrations to facilitate thegeneration of a standard curve to which the signal detected in the testsample can be compared. Alternatively, a standard curve can be generatedby preparing dilutions of a single humanized monoclonal antibodysolution provided in the kit.

Kits according to the present disclosure can include a solid phasecapable of binding autoantibodies present in the test sample, a firstantibody that binds to at least one epitope on the analyte of interest,the first antibody bound to the solid phase, a second antibody thatbinds to at least one epitope on the analyte of interest, andinstructions for detecting or quantifying the analyte of interest. Incertain embodiments test kits according to the present disclosure mayinclude the solid phase as a material such as a magnetic particle, abead, a test tube, a microtiter plate, a cuvette, a membrane, ascaffolding molecule, a quartz crystal, a film, a filter paper, a discor a chip.

Test kits according to the present disclosure can include for examplenon-human monoclonal antibodies against the analyte of interest, as thefirst and second antibodies. The kit may also include a detectable labelthat can be or is conjugated to the second antibody. In certainembodiments, the test kit includes at least one direct label, which maybe an enzyme, oligonucleotide, nanoparticle chemiluminophore,fluorophore, fluorescence quencher, chemiluminescence quencher, orbiotin. In some embodiments, the direct label is an acridinium compoundsuch as an acridinium-9-carboxamide according to formula I:

wherein R1 and R2 are each independently selected from the groupconsisting of: alkenyl, alkynyl, aryl or aralkyl, sulfoalkylcarboxyalkyl and oxoalkyl, and

wherein R3 through R15 are each independently selected from the groupconsisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and

optionally, if present, X^(θ) is an anion.

Alternatively, the acridinium compound can be anacridinium-9-carboxylate aryl ester having a structure according toformula II:

wherein R1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,carboxyalkyl and oxoalkyl; and

wherein R3 through R15 are each independently selected from the groupconsisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and

optionally, if present, X^(θ) is an anion.

Test kits according to the present disclosure and which include anacridinium compound can also include a basic solution. For example, thebasic solution can be a solution having a of at least about 10.

In certain embodiments, test kits according to the present disclosuremay further include a hydrogen peroxide source, such as a buffersolution, a solution containing hydrogen peroxide, or a hydrogenperoxide generating enzyme. For example, test kits may include an amountof a hydrogen peroxide generating enzymes selected from the following:(R)-6-hydroxynicotine oxidase, (S)-2-hydroxy acid oxidase,(S)-6-hydroxynicotine oxidase, 3-aci-nitropropanoate oxidase,3-hydroxyanthranilate oxidase, 4-hydroxymandelate oxidase,6-hydroxynicotinate dehydrogenase, abscisic-aldehyde oxidase, acyl-CoAoxidase, alcohol oxidase, aldehyde oxidase, amine oxidase, amine oxidase(copper-containing), amine oxidase (flavin-containing), aryl-alcoholoxidase, aryl-aldehyde oxidase, catechol oxidase, cholesterol oxidase,choline oxidase, columbamine oxidase, cyclohexylamine oxidase,cytochrome c oxidase, D-amino-acid oxidase, D-arabinono-1,4-lactoneoxidase, D-arabinono-1,4-lactone oxidase, D-aspartate oxidase,D-glutamate oxidase, D-glutamate (D-aspartate) oxidase,dihydrobenzophenanthridine oxidase, dihydroorotate oxidase,dihydrouracil oxidase, dimethylglycine oxidase, D-mannitol oxidase,ecdysone oxidase, ethanolamine oxidase, galactose oxidase, glucoseoxidase, glutathione oxidase, glycerol-3-phosphate oxidase, glycineoxidase, glyoxylate oxidase, hexose oxidase, hydroxyphytanate oxidase,indole-3-acetaldehyde oxidase, lactic acid oxidase, L-amino-acidoxidase, L-aspartate oxidase, L-galactonolactone oxidase, L-glutamateoxidase, L-gulonolactone oxidase, L-lysine 6-oxidase, L-lysine oxidase,long-chain-alcohol oxidase, L-pipecolate oxidase, L-sorbose oxidase,malate oxidase, methanethiol oxidase, monoamino acid oxidase,N6-methyl-lysine oxidase, N-acylhexosamine oxidase, NAD(P)H oxidase,nitroalkane oxidase, N-methyl-L-amino-acid oxidase, nucleoside oxidase,oxalate oxidase, polyamine oxidase, polyphenol oxidase,polyvinyl-alcohol oxidase, prenylcysteine oxidase, protein-lysine6-oxidase, putrescine oxidase, pyranose oxidase, pyridoxal 5′-phosphatesynthase, pyridoxine 4-oxidase, pyrroloquinoline-quinone synthase,pyruvate oxidase, pyruvate oxidase (CoA-acetylating), reticulineoxidase, retinal oxidase, rifamycin-B oxidase, sarcosine oxidase,secondary-alcohol oxidase, sulfite oxidase, superoxide dismutase,superoxide reductase, tetrahydroberberine oxidase, thiamine oxidase,tryptophan α,β-oxidase, urate oxidase (uricase, uric acid oxidase),oxidase, xanthine oxidase, oxidase and combinations thereof.

In certain embodiments, test kits according to the present disclosureare configured for detection or quantification of one of the followingspecific analytes of interest cardiac troponin, thyroid stimulatinghormone (TSH), beta human chorionic gonadotropin (beta-HCG);myeloperoxidase (MPO), prostate specific antigen (PSA), human B-typenatriuretic peptide (BNP), myosin tight chain 2, myosin-6 and myosin-7.In such embodiments, the test kits include a first antibody and a secondantibody that each bind to an epitope on the selected analyte ofinterest, i.e. a first antibody and a second antibody and secondantibody that each bind to an epitope on one of the following: cardiactroponin, thyroid stimulating hormone (TSH), beta human chorionicgonadotropin (beta-HCG); myeloperoxidase (MPO), prostate specificantigen (PSA), human B-type natriuretic peptide (BNP), myosin lightchain 2, myosin-6 and myosin-7.

Test kits according to the present disclosure preferably includeinstructions for carrying out one or more of the immunoassays of theinvention. Instructions included in kits of the present disclosure canbe affixed to packaging material or can be included as a package insert.While the instructions are typically written or printed materials theyare not limited to such. Any medium capable of storing such instructionsand communicating them to an end user is contemplated by thisdisclosure. Such media include, but are not limited to, electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. As used herein, the term“instructions” can include the address of an hamlet site that providesthe instructions.

D. Adaptations of the Methods of the Present Disclosure

The present disclosure is for example applicable to the jointly ownedcommercial Abbott Point of Care (i-STAT™) electrochemical immunoassaysystem which performs sandwich immunoassays for several cardiac markers,including TnI, CKMB and BNP. Immunosensors and ways of operating them insingle-use test devices are described in jointly owned Publication Nos.US 20030170881, US 20040018577, US 20050054078, and US 20060160164, eachof which is incorporated herein by reference. Additional background onthe manufacture electrochemical and other types of immunosensors isfound in jointly owned U.S. Pat. No. 5,063,081 which is alsoincorporated by reference.

By way of example, and not of limitation, examples of the presentdisclosures shall now be given.

Example 1 Capture of Human IgG on a Magnetic Conjugated to an AnalyteSpecific IgG. (ELN ref: E000777-281)

Microparticles: Carboxylated magnetic microparticles (PolymerLaboratories), MES buffer, EDAC, and anti-troponin-I purified murineIgG's (8E10 and MO6) were added together and the suspension was mixed.The particles were washed with 1% Tween in PBS, then 1% BSA in PBS, thenwith a diluent (normal goat IgG 0.028 L/L; TRIS 6.05 g/L; EGTA 9.51 g/L;sodium chloride; 5.8 g/L; BSA 10.0 g/L; Brij 35 3.0 g/L; sodium alkylparaben 1.0 g/L; sarafloxacin hydrochloride 0.01 L/L; Tectronic 130710.0 g/L; sucrose 136 g/L), and adjusted to a final concentration of 1%solids.

Human IgG stock solution: Human IgG (Sigma cat#140506 lot 0471K7635) wasdiluted in PBS to give a solution of 50 mg/mL.

Human IgG standard solutions: The human IgG stock solution was seriallydiluted in PBS to give standard solutions at 25, 12.5, 6.25, 3.13, 1.57,0.78, 0.39, 0.20, 0.10, 0.05, and 0.025 mg/mL.

Anti-human IgG detection conjugate: An anti-human IgGacridinium-9-carboxamide-labeled conjugate solution (25 ng/mL) wasprepared in MOPS buffer (pH 6.3, 0.05% BSA, 1% Triton, 0.1% dextransulfate).

Protocol: The human IgG standard solutions were assayed in duplicate onan ARCHITECT i2000SR using the ARCHITECT stat-Troponin-I protocol, andthe microparticles and detection conjugate described above. Apoint-to-point dose-response curve was constructed by plotting the RLUresponse obtained from the ARCHITECT assay versus the human IgGconcentration tested as shown graphically in FIG. 3.

Example 2 Assay for Human IgG in Normal Serum on a MagneticMicroparticle Conjugated to an Analyte Specific IgG. (ELN ref:E000777-281)

A normal serum sample was serially diluted in PBS and analyzed using thereagents and protocol from Example 1. The RLU response from the assaywas plotted versus the human IgG concentration calculated from thedose-response curve in Example 1 as shown graphically in FIG. 4.

Example 3 Effect of Captured Human Anti-Cardiac Troponin-1 on a CardiacTroponin-I Immunoassay. (ELN ref: E000777-278)

Two samples were chosen from a population of normal blood donorsscreened for anti cardiac troponin-I autoantibodies (U.S. Ser. No.11/588,073); one was determined to have low-reactivity (LR) in the assayfor while the other had high reactivity (HR). Cardiac troponin-1(BiosPacific cat# J34170359) was added to aliquots of each sample at twoconcentrations to give final cTnI concentrations of 0.25 and 1.5 ng/mL.Each sample was analyzed using the microparticles described in Example 1and cardiac troponin-I specific detection conjugate and diluentssupplied in the ARCHITECT Stat Troponin-I Kit (cat#2K41-30). The samplecontaining a high level of autoantibodies reactive with cardiactroponin-I showed an increased sensitivity to cardiac troponin-I at boththe 0.25 and 1.5 ng/mL level. This is reflected in a 36-37% increase inthe B/A and C/A ratios in the HR sample relative to the LR samples,which had only a very low level of autoantibodies reactive with cardiactroponin-I (Table 2),

TABLE 2 Increased sensitivity to cardiac troponin-I in the presence ofcaptured cTnI-autoantibodies cTnI Sample (ng/mL) RLU RLU/A % Inc HR A 0327 — — B 0.25 740 B/A 2.3 37% C 1.5 999 C/A 3.1 36% LR A 0 402 — — — B0.25 662 B/A 1.6 — C 1.5 906 C/A 2.3 —

One skilled in the art would readily appreciate that the immunoassaysdescribed in the present disclosure are well adapted to carry out theobjects and obtain the ends and advantages mentioned, as well as thoseinherent therein. The molecular complexes and the methods, procedures,treatments, molecules, specific compounds described herein are presentlyrepresentative of preferred embodiments, are exemplary, and are notintended as limitations on the scope of the invention. It will bereadily apparent to one skilled in the art that varying substitutionsand modifications may be made to the present disclosure disclosed hereinwithout departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which thepresent disclosure pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The present disclosure illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising,”“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the present disclosure claimed. Thus, itshould be understood that although the present disclosure has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

1. An immunodetection composition comprising: a) a first detectioncomplex comprising a first antibody reactive with an analyte of interestand bound to a solid phase, the analyte of interest, and a secondantibody reactive with the analyte of interest, wherein the secondantibody has a detectable label; and b) a second detection complexcomprising an autoantibody reactive with the analyte of interest andbound to the solid phase, the analyte of interest, and the secondantibody, wherein the first and second complexes generate a measurableoptical, electrical, or change-of-state signal from the detectablelabel.
 2. The composition according to claim 1, wherein the firstdetection complex is bound to the second detection complex on the solidphase.
 3. The composition of claim 1, wherein the second antibody isconjugated to a detectable label, wherein the detectable label is anenzyme, oligonucleotide, nanoparticle chemiluminophore, fluorophore,fluorescence quencher, chemiluminescence quencher, or biotin.
 4. Thecomposition of claim 1 or 2, wherein the optical signal is measured asan analyte concentration dependent change in chemiluminescence,fluorescence, phosphorescence, electrochemiluminescence, ultravioletabsorption, visible absorption, infrared absorption, refraction, surfaceplasmon resonance.
 5. The composition of claim 1 or 2, wherein theelectrical signal is measured as an analyte concentration dependentchange in current, resistance, potential, mass to charge ratio, or ioncount.
 6. The composition of claim 1 or 2, wherein the change-of-statesignal is measured as an analyte concentration dependent change in size,solubility, mass, or resonance.
 7. The composition of claim 1, whereinthe analyte of interest is a cardiac troponin, thyroid stimulatinghormone (TSH), beta human chorionic gonadotropin (beta-HCG);myeloperoxidase (MPO), prostate specific antigen (PSA), human B-typenatriuretic peptide (BNP), myosin light chain 2, myosin-6 or myosin-7.8. The composition of claim 1, wherein the test sample is whole blood,serum, plasma.
 9. The composition of claim 3, wherein the detectablelabel is an acridinium-9-carboxamide having a structure according toformula I:

wherein R¹ and R² are each independently selected from the groupconsisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,carboxyalkyl and oxoalkyl, and wherein R³ through R¹⁵ are eachindependently selected from the group consisting of: hydrogen, alkyl,alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl,hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,carboxyalkyl and oxoalkyl; and optionally, if present, X^(Θ) is ananion.
 10. The composition of claim 3, wherein the detectable label isan acridinium-9-carboxylate aryl ester having a structure according toformula II:

wherein R¹ is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,carboxyalkyl and oxoalkyl; and wherein R³ through R¹⁵ are eachindependently selected from the group consisting of: hydrogen, alkyl,alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl,hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl,carboxyalkyl and oxoalkyl; and optionally, if present, X^(Θ) is ananion.
 11. The composition of claim 1, wherein the solid phase isselected from the group consisting of a magnetic particle, bead, testtube, microtiter plate, cuvette, membrane, a scaffolding molecule,quartz crystal, film, filter paper, disc and chip.
 12. The compositionof claim 1, wherein the first antibody is selected from the groupconsisting of a polyclonal antibody, a monoclonal antibody, a chimericantibody, a human antibody, and an affinity maturated antibody.
 13. Thecomposition of claim 1, wherein the second antibody is selected from thegroup consisting of a polyclonal antibody, a monoclonal antibody, achimeric antibody, a human antibody, and an affinity maturated antibody.